Discovery and Validation of a Novel Step Catalyzed by OsF3H in the Flavonoid Biosynthesis Pathway

Biology

Sanjita Paudel¹  Sangkyu Lee¹  Lubna²  Kyung-Min Kim³  Rahmatullah Jan³  Sajjad Asaf⁴ 

[1] College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu 41566, Korea;Department of Botany, Garden Campus, Abdul Wali Khan University, Mardan 23200, Pakistan;Division of Plant Biosciences, School of Applied Biosciences, College of Agriculture & Life Science, Kyungpook National University, 80 Dahak-ro, Buk-gu, Daegu 41566, Korea;Natural and Medical Science Research Center, University of Nizwa 616, Nizwa 611, Oman;

关键词: kaempferol;    naringenin;    nuclear magnetic resonance;    yeast episomal plasmid;    hydroxylation;   

DOI  :  10.3390/biology10010032

来源: DOAJ

【 摘 要 】

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. OsF3H was cloned into pRS42K yeast episomal plasmid (YEp) vector and the activity of the target gene was analyzed in engineered and empty strains. We confirmed a novel step of kaempferol and quercetin biosynthesis directly from naringenin, catalyzed by the rice flavanone 3-hydroxylase (F3H). The results were confirmed through thin layer chromatography (TLC) followed by western blotting, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry LCMS-MS. TLC showed positive results when comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed the lack of OsF3H activity in empty strains and confirmed high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for the conversion of naringenin to both kaempferol and quercetin.

【 授权许可】

Unknown